We have been studying crystallins, the abundant water-soluble proteins responsible for the optical properties of the eye lens as a model of tissue-specific gene expression. In FY2001 we have examined the mouse small heat shock protein alphaB-crystallin gene. An alphaB-crystallin-related gene (HSPB2) is present 1 kb upstream of the mouse authentic alphaB-crystallin gene and transcribed in the opposite direction. Interestingly, this alphaB-related gene is expressed in muscle and heart, but negligibly in lens. We have shown by placing either firefly or Renilla luciferase reporter genes at each end of the linked promoters that the upstream aB-crystallin enhancer affects the authentic alphaB-crystallin gene in an orientation-specific manner. The enhancer has little influence on the HSPB2 promoter. The possibility that the unidirectional role of the alphaB enhancer is due to an insulator is being tested. In a collaborative project with Dr. Eviatar Nevo (University of Haifa, Israel) we have shown that the alphaB-crystallin promoter/enhancer of the mouse and blind mole rat are very similar but not identical. Despite their similarity the mole-rat and the mouse alphaB-crystallin promoter/enhancer-luciferase transgenes differ in that both are expressed in skeletal muscle and heart, but only the mouse transgene is expressed to any significant extent in the mature lens of transgenic mice. Recently we have shown that the mole rat promoter functions during early embryonic in the transgenic mouse, but turns off later in development, suggesting the existence of a developmental switch for alphaB-crystallin gene expression. Three additional projects were completed this year. One showed that Prox1, a homeodomain transcription factor, redistributes from the cytoplasm to the nucleus during lens fiber cell differentiation and activates a number of crystalllin promoters. Another demonstrated that lens-specificity was convserved in the betaB1-crystallin promoter of chicken and mice. The third demonstrated that AP2alpha is a negative regulator of terminal fiber cell differentiation, possibly through the maintenance of cell-cell and/or cell-matrix adhesion of lens epithelial cells. Previously we discovered novel J1, J2 and J3-crystallins in the jellyfish lens. Now we matched J1-crystallins with an EST from zebrafish brain and showed that J3-crystallin is similar to saposins. In addition we showed that the scallop crystallin, called omega-crystallin, is an inactive dimeric aldehyde dehydrogenase. We also have provided evidence for the conservation of crystallin gene regulation throughout vertebrates and invertebrates by showing that the scallop ALDH/omega-crystallin promoter contains potential cis-control elements remarkably similar to those governing lens-specific activity of the mouse and chicken alphaA-crystallin promoters. Among these are overlapping Pax6 and CREB sites that bind their respective factors and appear functional in mutagenesis and transfection experiments.